Introduction

Scarlet fever is a classic exanthem of childhood caused by the bacterium Streptococcus pyogenes (group A streptococcus) which was associated with frequent life-harm among human children until the early 1900s.

1

Developed microbes and recurrent streptococcal disease.

In the early 1

900s, well before the widespread use of antibiotics, the occurrence and severity of scarlet fever began, a phenomenon that remains largely inexplicable.

2

Duncan SR

Scott S

Duncan CJ

Modeling the dynamics of scarlet fever epidemics during the 19th century.

A potential (unstable) hypothesis is that the streptococcal bacteria that caused the disease may have undergone a pathogenetic change that led to a reduction in the invasive and septic effects of scarlet fever.

Since the 1940s, scarlet fever has followed a seasonal spring pat tern – which peaked between March and May while less frequent during the rest of the year – without the major cyclical epidemics observed in the early 1900s.

Surgery in invasive infections can regularly follow a similar seasonal pattern for reasons that are incompletely understood. In 2014, England had an unexpectedly sharp increase in infections with scarlet fever, with over 15,000 notifications of illnesses – a marked increase in prevalence compared to previous decades.

the increase in infections was not associated with any increase in the incidence of invasive disease. Even greater seasonal increases in scarlet fever were observed in 2015, when there were over 17,000 notifications, and in 2016, when there were over 19,000 notifications.

In spring 2016, an increase of 1-5 times the number of laboratory-confirmed invasive S pyogenes infections compared to those in the previous 5 years coincided with the peak in reports of scarlet fever.

led us to speculate that the relationship between scarlet fever and invasive disease in 2016 could be dependent on strain.

Research in context

Evidence before this study

In March to May 2016, an unexpected increase was seen in reports of invasive Streptococcus pyogenes infections in England, coinciding with a national increase in seasonal bald fever notifications (child exanthema also caused by S pyogenes ). Since 2014, reports of scarlet fever in England have reached unexpectedly high levels and peaked between March and May each year, although reports of invasive S pyogenes infections in 2014 were within expected limits, in contrast to 2016. We aimed at test the hypothesis that the link between scarlet fever and invasive infection patterns may be stem-related and identified during the process of the emergence of a new M1T1 lineage. We searched PubMed for clinical and laboratory studies published before March 1, 2019, using the search terms "scarlet fever" and "upturn" or "mortality", as well as " emm 1" or "M1T1" and "streptococcus". We also searched with the terms "SpeA" or "scarlet fever toxin" or "erythrogenic toxin" and "streptococcus" and "regulation". We identified studies that described recent and historical trends in the occurrence of scarlet fever, studies that described trends in strain types that cause invasive infections, and studies that linked SpeA to dominant strains. We also found studies of toxin expression that reported links to phage induction, growth phase regulation, transcriptional regulators, proteolysis, and host proteins as potential regulators.

Added value for this study

Our study provides a molecular explanation for the association between increased incidence of scarlet fever and increased incidence of invasive S. pyogenesis infections, by identifying an emerging lineage of M1T1 pyogenes (M1 UK ) which expanded rapidly to become the largest single contributor to both non-invasive and invasive infections in 2016. The results give rise to the possibility of historical links between epidemic waves of scarlet fever and invasive infections as well may have been linked to stem pathogenicity, in addition to the general public's susceptibility. Genomic analysis confirmed that the strains that cause scarlet fever do not differ from those that cause streptococcal pharyngitis and rare invasive infections. Increases in a disease can lead to increases in everyone, especially if this line is very pathogenic. The growing line was characterized by a number of genetic changes that were predictable for increased production of SpeA, and this increased production was confirmed by laboratory tests. While this may just be one of many changes in the new line, increased production of SpeA is predicted to improve bacterial fitness, which is suggested by the increasing dominance of the new line compared to older M1T1 strains in England. The work emphasizes that streptococcal families in group A may differ in pathogenicity.

Implications of all available evidence

Notices of scarlet fever in England during the period 2014-18 are the highest seen since 1960, and the incidence of young children exceeds that reported in other countries. It is uncertain whether the increase in scarlet fever is a result of practice change or other population or environmental factors; the new descent described is not involved in the initial ascent. However, there is a need to limit the rising trend. Efforts directed at the vulnerable population have the potential to reduce the reservoir of S pyogenes which can seed more harmful invasive infections. Research to identify the most appropriate intervention is in progress and practical guidelines for streptococcal pharyngitis may need to take into account stress variation and wider population effects. Increased S pyogenes' disease activity could provide a platform for tribal development and expansion, which highlights an unforeseen consequence of modern epidemics. The genetic changes in the emerging M1T1 clone require detailed laboratory research to understand the broader phenotypic changes that have occurred and their molecular basis, including transmissibility and response to treatment. It is not known whether the new line will diminish over time, as other lines have done, or whether it will remain dominant in the population, and monitoring is needed. The discovery of new genealogical representatives outside the UK underlines the need for global surveillance and increased vigilance if strains with increased fitness and altered phenotype are detected.

Microbiological monitoring of S pyogenesis upper respiratory tract infections in England is limited, as most doctors do not routinely take samples for bacteriological diagnosis of sore throat. However, all isolated S-pyogenes cultured from samples submitted from the population in north-west London are collected and archived by Imperial College Infection Biobank, which allows longitudinal study of strains causing both non-invasive and invasive infections. Together with the National Reference Laboratory, which systematically archives invasive S pyogenes isolates from all over the country, the collection provides unique insights into the relationship between upper respiratory tract isolates and isolates from rare, but severe and invasive infections.

S pyogenes can be serotyped or genotyped on the basis of the M antigen encoded by the em gene. Changes in disease incidence are sometimes characterized by the expansion of specific em genotypes.

Using a combination of epidemiological and bacteriological approaches, we set out to identify em genotypes responsible for S pyogenesis infections during the 2014–16 season scarlet fever annually, then expanded nationally to identify bacterial determinants that may explain the observed increase in invasive disease due to S pyogenesis 2016.

Results [19659002] The population of north-west London had an annual increase in scarlet fever notifications between 2014 and 2016, which was representative of the country as a whole compared to national notification data (Figure 1A). We also compared notifications of invasive S pyogenes infections in north-west London with national data (Appendix p 2), which showed a marked increase in S pyogenes invasive disease 2016 in north-west London during that season with scarlet fever that also reflected the national pattern for increased reporting in 2016.

View full caption

(A) Monthly scarlet fever in north-west London (bars) 2013–16, showing the increase in notifications between March and May, which peaked in 2016. National scarlet fever messages (dashed lines) are shown for comparison. (B) em genotyping of all upper airway isolates from S pyogenes from north-west London between March and May each year during 2014-16. em 1 strains emerged as the dominant upper respiratory genotype after 2016.

emm genotypes of all S pyogenes upper airway isolates obtained in northwestern London during the 2015 and 2016 scarce fever seasons were determined and compared with existing data from 2014.

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Turner CE

Pyzio M

Song B

et al.

Scarlet fever increases in England and molecular genetic analysis in north west London, 2014.

To identify any genetic basis for the expansion of em 1 S pyogenes among the upper respiratory tract isolates collected in London, the genomes of all non-invasive em 1 isolates available from north-west London from 2009 to 2016 were sequenced (n = 135). Single nucleotide polymorphism (SNP) -based analysis of em 1 strains pointed to the emergence of a new em 1 lineage (designated M1 United Kingdom ), which could be differentiated from the contemporary emm 1 population (M1 global ) through the presence of 27 nuclear SNPs in regulatory and metabolic genes (Figure 2, Appendix p 7). The earliest member of M1 UK descent was identified in 2010 and five intermediate isolates (with 13 or 23 of the unique SNPs) were detected between 2009 and 2012 (Appendix p 3). Similar to M1 global M1 UK was isolated from all age groups, but included more cases of scarlet fever and recent transmission evidence than M1 global (Appendix p 3 ). From 2015 onwards, about two-thirds of non-invasive em were 1 isolate from north-west London within the new M1 UK descent (22 [71%] of 31 isolates 2015 and 30 [65%] of 46 2016). Recombination and pan-genome analysis provided no evidence of gain or loss of transmissible elements when comparing M1 global and M1 UK strains. Line-specific acquisition of antimicrobial resistance genes was not detected; evidence of mefA and msrD macrolide resistance locus was found in only one M1 UK isolate, while eight M1 global isolates contained antimicrobial resistance genes (one isolate with mefA and msrD locus, one with the tetracycline resistance gene tetM and six with the aminoglycoside resistance gene [19459008

Scarlet fever is a toxin-mediated syndrome historically associated with the expression of the phage-encoded erythrogenic toxin SpeA, [1965900319659105] Nelson K

Schlievert PM

Selander RK

Musser JM

Characterization and clone distribution of four alleles of the speA gene encoding pyrogenic exotoxin A (scarlet fetal toxin) in [19459Streptococcuspyogenes.

The gene that is possessed by all em 1 isolates studied in this study. Among the 27 M1 UK delimiting SNPs, three non-synonymous mutations were identified in the gene rofA encoding the free-standing transcriptional regulator RofA,

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Fogg GC [19659008] Gibson CM

Caparon MG

Identification of rofA a positive-acting regulatory component of prtF expression: use of a m gamma delta-based shuttle mutagenesis strategy in Streptesococ py .

which, together with a homologue, nra has been implicated as a repressor for SpeA production in some, but not all, streptococcal genotypes.

Beckert S

Kreikemeyer B

Podbielski A

Group A streptococcal rofA gene is involved in the control of multiple virulence genes and eukaryotic cell binding and internalization.

16

Molinari G

Rohde M [19659008] Talay SR

Chhatwal GS

Beckert S

Podbielski A

Den role played by group A streptococcal negative regulator Nra on bacterial interactions with epithelial cells.

The sequence of speA in non-invasive M1 UK and M1 global isolates was identical to the reference strain MGAS5005. Integration sites for the phage transporting speA in sequenced non-invasive M1 UK and M1 global isolations were consistent with those in the reference strain and mapped sequences from M1 UK and M1 global isolates were very similar (if not identical) to the reference stem phage 5005.1; no evidence of translocated superantigens or duplication of speA .

Although there was no increase in reports of invasive disease in 2014, the first year with increased scarlet fever activity, there was a significant increase nationally in spring 2016 compared with the same period (March to May) in the previous three years (tax rate 1 · 43 [95% CI 1·31–1·56] appendix p 2). Although the number of invasive diseases in north-west London was modest when taken into account on a monthly basis compared to national data (Appendix p 2), the seasonal increase in notifications observed nationally matched. EM genotyping of all invasive disease isolates referred to the National Laboratory showed significant absolute and relative year-on-year increases EM 1, from 183 (31%) of 587 isolates between March and May 2015 , to 267 (42%) of 637 during the same period 2016 (p vs 2015 values), which peaked in March 2016 (Figure 4A).

Figure 4 Prevalence of em 1 strains among invasive Streptococcus pyogenes infections nationally during scarlet fever seasons and the emergence of M1 ] descent over time

View full captions

(A) em genotypes of invasive S pyogenes isolates referred to national reference laboratory per month during 2014-16. Increases in total invasive disease cases were observed locally and nationally during March to May 2016 (Appendix p 2). (B) Maximum Likelihood Phylogenetic Tree Constructed by Nucleotide Nucleotide Polymorphisms (Excluding Prophage Regions) of Genome-Sequenced Invasive Em 1 S pyogenes Isolate in England and Wales (n = 552 ) between March and May each year during 2013-16. Shading in gray indicates the emerging line M1 UK . Clustering was not observed based on geographical origin, indicating the emergence of genus at national level. The black star indicates the reference strain MGAS5005. The scale bar represents the number of nucleotide substitutions per site.

To determine whether invasive S pyogenes infections can be affected by the emerging M1 UK descent, we compared the genome sequences for 552 invasive em 1 isolates (Appendix pp 12–27) in England and Wales from March to May each year in 2013–16. Focus first on London, where sequence data from non-invasive isolates were available for comparison, SNP-based phylogenetic analysis showed mixing of the 31 sterile site invasive and 135 non-invasive isolates; 16 (84%) of 19 invasive strains obtained in 2015 and 2016 were in the emerging M1 UK lineage, compared to only five (42%) of 12 obtained between 2013 and 2014 (Appendix p 4). Four (13%) of 31 invasive isolates were identical to, or no more than two SNPs different from non-invasive isolates in the community, consistent with the most recent transmission.

We then analyzed the genome sequences for all 552 em 1 sterile site isolates collected between March and May each year from 2013 to 2016, obtained nationally from different geographical locations in England and Wales. 425 (77%) of 552 invasive emm 1 strains existed within the new line (Figure 4B), which was present in the UK's invasive isolate population as early as 2013. Like M1 global strains, M1 UK strains differed phylogenetically from historic Britain em 1 scarlet fever speA -positive isolate NCTC8198 and em speC -positive SF370 (Appendix s 5). Longitudinal analysis of all available 1240 UK em 1 sequences (Appendix p 28)

6

Kapatai G

Coelho J

Platt S

Chalker VJ

All through sequencing of group A streptococci: development and evaluation of an automated pipeline for emm gene typing.

Community outbreaks of group A streptococci revealed through follow-up.

obtained from invasive and non-invasive disease cases showed an annual increase of M1 UK so that in 2016, M1 UK strains represented more than 80% of all available em 1 isolate in the UK, over M1 global strains (Figure 5A).

(A) Fractions of M1 UK and M1 global isolates among total sequence-determined invasive and non-invasive em 1 S pyogenes isolate (n = 1240) annually in the UK between 2007 and 2016. (B) M1 UK descent in a global context. Maximum likelihood of phylogenetic trees constructed from SNP cores (excluding prophage regions) comparing all sequenced Great Britain em 1 isolate to global em 1 populations from North America, Nordic countries and Asia (n = 2800 isolate). Shading in gray indicates the emerging line M1 UK ; orange arc indicates intermediate isolates located outside M1 UK but has 13 or more of the 27 SNPs found in M1 UK including three SNPs in rofA . UK and international emm 1 isolate occurs throughout the tree, but isolate within M1 UK is exclusively from the UK, except two individual isolates from Denmark and the USA (arrows). The scale bar indicates the number of nucleotide substitutions per site. See Appendix (p. 6) for the unrooted tree. SNPs = single-nucleotide polymorphisms.

Phylogenetic Comparison of Great Britain em 1 sequences with available international sequences from North America, Nordic regions, United Kingdom and Southeast Asia (Appendix p 28)

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Chochua S

Metcalf BJ

Li Z

et al.

Population and whole through sequence-based characterization of invasive group A streptococci recovered in the United States in 2015.

Diskussion

Severe group A streptococcal infections associated with a toxic shock-like syndrome and scarlet fever toxin A.

and subsequently dominated by the emm1 lineage.

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Nasser W

Beres SB

Olsen RJ

et al.

Evolutionary pathway to increased virulence and epidemic group A streptococcus disease derived from 3,615 genome sequences.

In this study, we showed that the originally poly clonal upsurge of scarlet fever in England has more recently been characterised by the emergence of a new emm1 S pyogenes lineage that produces significantly higher levels of SpeA than other contemporary emm1 strains. The new M1UK lineage showed an apparent fitness advantage within the population, manifest during the scarlet fever seasons of 2015 and 2016. Phylogenetic analysis showed the emergent lineage to be the dominant cause of invasive S pyogenes infections in England in 2016, and indicated that isolates from symptomatic throat infections and scarlet fever represent the major reservoir for invasive infections. The data support the hypothesis that transmission of virulent emm1 strains with enhanced ability to cause scarlet fever could underlie the contemporaneous rise in invasive S pyogenes disease.

An unprecedented year-on-year increase in scarlet fever notifications, the underlying basis for which remains unclear,

National Institute for Health and Care ExcellenceRespiratory tract infections—antibiotic prescribing. Prescribing antibiotics for self-limiting respiratory tract infections in adults and children in primary care.

although causal links have not been established. One unforeseen consequence of medical practice change was that the capacity to investigate such an upsurge was undermined by a reduction in diagnostic testing at the national level. In northwest London, however, where strains are collected for epidemiological analysis, a significant increase in genotype emm4 pharyngitis strains was observed during the 2014 upsurge in scarlet fever, while emm3 was the main genotype associated with physician-reported scarlet fever.

emm1 was infrequent in 2014, contrasting with the increase we observed between 2015 and 2016, when genotype emm1 S pyogenes became the dominant cause of upper respiratory tract infections regionally, and invasive disease notifications nationally.

Our genome sequence analysis revealed the emergence of a new emm1 lineage, separated from all other emm1 strains by 27 unique core SNPs, including three within a gene encoding a potential SpeA regulator, RofA. SpeA is usually the only phage-encoded superantigen in contemporary emm1 S pyogenesand has been implicated in the re-emergence of severe invasive S pyogenes infections in the 1980s.

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Nasser W

Beres SB

Olsen RJ

et al.

Evolutionary pathway to increased virulence and epidemic group A streptococcus disease derived from 3,615 genome sequences.

,

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Stevens DL

Tanner MH

Winship J

et al.

Severe group A streptococcal infections associated with a toxic shock-like syndrome and scarlet fever toxin A.

Although the roles of any specific SNPs were not investigated in the current study, we hypothesise that increased SpeA production by M1UK strains might be an important contributor to the apparent fitness of the new lineage within the nasopharynx. As a phage-encoded superantigen, SpeA is hypothesised to trigger scarlet fever in susceptible children, and has been shown to permit nasopharyngeal infection in humanised models of murine streptococcal infection, plus potential induction of immunity when administered as a toxoid.

Recurrent group A streptococcus tonsillitis is an immunosusceptibility disease involving antibody deficiency and aberrant TFH cells.

Thus, production of SpeA might augment the ability of S pyogenes to cause scarlet fever and paediatric pharyngitis. Whether population immunity to SpeA will lead to an eventual decline in the lineage will be of interest to monitor.

Griffiths type 1 S pyogenes strains

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The serological classification of Streptococcus pyogenes.

(later designated serotype M1; genotype emm1) were the first to be classically associated with scarlet fever in the early 20th century. Although the oldest emm1 scarlet fever reference strains dating from the 1920s possess and produce phage-encoded SpeA

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Sriskandan S

Moyes D

Buttery LK

et al.

Streptococcal pyrogenic exotoxin A release, distribution, and role in a murine model of fasciitis and multiorgan failure due to Streptococcus pyogenes.

emm1 strains circulating later in the mid-20th century lacked phage-encoded SpeA.

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Nasser W

Beres SB

Olsen RJ

et al.

Evolutionary pathway to increased virulence and epidemic group A streptococcus disease derived from 3,615 genome sequences.

The epidemic success of M1T1 clonal emm1 strains of S pyogenes that subsequently emerged in the 1980s is attributed to acquisition of a more active NADase–streptolysin O locus, as well as acquisition of phage- encoded speA2, an allele of speAand the DNAse-encoding gene sda.

10

Nasser W

Beres SB

Olsen RJ

et al.

Evolutionary pathway to increased virulence and epidemic group A streptococcus disease derived from 3,615 genome sequences.

It is, therefore, surprising that these epidemic emm1 strains, forming the M1global lineage, apparently produce very little SpeA in vitro. The three SNPs identified in the rofA gene might contribute to the greater abundance of SpeA produced by the emergent M1UK lineage strains, but appear insufficient alone to account for this change. Gene regulation could be affected by a number of the additional SNPs implicated. A shift to enhanced SpeA expression in the M1UK lineage might represent just one component of a new phase in the evolution of emm1, although we have not yet explored other aspects of bacterial fitness.

Since the late 2000s, increased scarlet fever notifications have been reported in China,

The M1UK lineage is distinct from emm1 sequences identified in Asia, where different, non-emm1 genotypes have been reported as upsurge-associated, and incidence in very young children does not approach that reported in England.

Sampling of the non-invasive reservoir of S pyogenes in our study was limited to a single region of England, and single seasons in 2014–16; thus, we cannot be certain about whether the non-invasive isolate emm genotype or M1UK proportions altered outside these periods, and the analysis might be skewed through inadvertent inclusion of isolates from outbreaks. The collection used in this study, however, is the only systematic longitudinal collection of non-invasive strains available, and spans 2009–16. None of the samples included was identified as outbreak-associated, although clinical details were imperfect.

Groups of isolates differing by two SNPs or less were identified among non-invasive M1UK isolates, suggesting recent transmission, whereas no such groups were observed among the M1global non-invasive isolates, albeit these were from 2009–2014 when emm1 isolates were much less frequently identified. Whether the findings reflect inadvertent sampling of small outbreaks or greater outbreak potential cannot be addressed using these data.

Regardless of the drawbacks of regional sampling, the detection of a new lineage and increase in invasive emm1 infections prompted sequencing of invasive emm1 isolates referred to the national reference laboratory, which confirmed emergence of the same lineage at a national level, and excluded any artefact introduced by regional sampling. Our phylogenetic analysis of invasive isolates focused on the same season, in order to understand a rise in invasive disease, but published genome-sequenced invasive strains from the UK outside these months also showed emergence of the same lineage. Although the new lineage outcompeted the contemporary emm1 strains that were sequenced during the period studied, we do not know whether the lineage&#39;s success will endure.

Genetic analyses were limited by the nature of high-throughput short-read sequencing data, which do not provide fully assembled genome sequences for each strain and the phages therein; other lineage-specific differences might yet be identified using different approaches. Our study also did not address the mechanistic basis for fitness in the new lineage or the molecular basis for increased SpeA expression; links to specific SNPs are by association only, and detailed experimental work is underway to address such questions.

Scarlet fever is a visible and readily recognisable manifestation of S pyogenes infection that affects children aged 4–6 years.

Importantly, however, the surges observed in scarlet fever are accompanied by seasonal increases in streptococcal sore throat and tonsillitis in the wider population, also predominantly among children.

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Royal College of General Practitioners Research & Surveillance CentreWeekly returns service an nual report 2016–2017.

Although we cannot rule out the possibility that the precursors (ie, intermediates) or founders of the M1UK lineage were imported, we speculate that a generalised increase in S pyogenes activity in the wider population—which coincided with England&#39;s scarlet fever upsurges—might have provided the conditions required for adaptation and expansion of emm1 S pyogenes. Whether the M1UK lineage will be suited to other environments is unknown; management of streptococcal sore throat differs greatly between countries,

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Chiappini E

Regoli M

Bonsignori F

et al.

Analysis of different recommendations from international guidelines for the management of acute pharyngitis in adults and children.

as do other important factors such as climate. We previously reported the emergence of a new emm89 lineage that had lost the capsule locus but gained an active NADase–streptolysin O locus, in addition to four other major recombination events.

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Turner CE

Abbott J

Lamagni T

et al.

Emergence of a new highly successful acapsular group A streptococcus clade of genotype emm89 in the United Kingdom.

This emm89 lineage has now been identified across several continents.

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Zhu L

Olsen RJ

Nasser W

et al.

A molecular trigger for intercontinental epidemics of group A streptococcus.

The identification of two members of the new M1UK lineage among isolates outside the UK underlines the potential of such lineages to spread globally. Compared with other genotypes, emm1 S pyogenes has a recognised, heightened association with invasive infections.

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Chochua S

Metcalf BJ

Li Z

et al.

Population and whole genome sequence based characterization of invasive group A streptococci recovered in the United States during 2015.

The expansion of such a lineage within the community reservoir of S pyogenes might be sufficient to explain England&#39;s recent increase in invasive infection. Further research to assess the likely effects of M1UK on infection transmissibility, treatment response, disease burden, and severity is required, coupled with consideration of public health interventions to limit transmission where appropriate. Wider national and global surveillance will provide clearer understanding of the lineage&#39;s geographical reach and longer-term fitness, and permit enhanced public health readiness where necessary.

SS, EJ, NNL, CET, VC, and TL contributed to the conception of this project. HKL, XZ, MM, MP, MA, LL, EJ, and JP were responsible for collection of laboratory and genomic data. JYC, TL, and VC were responsible for epidemiological data. EJ and NNL interpreted and analysed data. EJ and CET undertook bioinformatics analysis of whole genome sequence data. SS, NNL, and EJ prepared the manuscript; NNL and EJ contributed equally. All authors contributed to the interpretation of results and critical review of the manuscript.

JP is a consultant to Next Gen Diagnostics LLC. All other authors declare no competing interests.